Novel endos mutant enzyme

ABSTRACT

The present invention provides an EndoS mutant enzyme having an amino acid sequence of EndoS D233Q and further having a particular additional mutation and exhibiting a reduced hydrolysis activity, in comparison with the activity of EndoS D233Q, to an N-linked sugar chain (N297-linked sugar chain) linked to Asn at position 297 in IgG and a gene encoding the same.

TECHNICAL FIELD

The present invention relates to a novel mutant ofendo-β-N-acetylglucosaminidase S (EndoS), a gene encoding the mutant, arecombinant plasmid, a transformant transformed with the plasmid, and amethod for modifying a sugar chain of an antibody using the mutant.

BACKGROUND ART

Glycoproteins widely reside in tissue in animals and plants, the cellmembrane and wall of eucaryotic microorganisms, and the like. Recently,it has been becoming clear that sugar chains of glycoproteins playimportant roles in mechanisms such as differentiation of cells,canceration, and cell-cell recognition and studies are underway on thecorrelation between the structure and the function of sugar chains toelucidate these mechanisms. In culture cell lines, a glycoprotein istranslated as a protein consisting of a uniform amino acid sequence andthen glycosylated by posttranslational modification to be delivered intothe culture medium. Since this posttranslational modification is notdetermined uniquely according to its amino acid sequence, sugar chainsthat are added vary in their structures even on the same proteinexpressed in the same culture system.

Antibodies are glycoprotein molecules having N-linked sugar chains(N297-linked sugar chains) linked to the Asn side chains at position 297located in the Fc regions of the heavy chain molecules. Antibodies areimportant molecules in basic studies and in the field of medicine andare eagerly studied and developed particularly as antibody medicines. Assuch, various effects of sugar chains are becoming clear (Non PatentLiterature 1: J. N. Arnold, et al., Annu. Rev. Immunol, 2007, 25,21050). Currently, the therapeutic antibodies that are used are mainlyof the IgG class of molecules. Such antibodies are produced by culturedanimal cells such as by CHO cells and NS0 cells in general andN297-linked sugar chains of antibodies produced by these animal cellsare biantennary complex-type sugar chains, which are heterogeneous inthe core fucose, the terminal sialyl and galactosyl groups, andbisecting GlcNAc (Non Patent Literature 2: R. Jefferis, Biotechnol.Prog., 2005, 21-11-6). It has been revealed that N297-linked sugarchains of antibodies have a large influence on effector activitiesincluding ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC(Non Patent Literature 3: Nimmerjahn, Nat. Rev. Immunol. 2008, 8, 34-47;Non Patent Literature 4: Jefferis Nat. Rev. Drug Discovery, 2009, 8,226-234) and the possibility that they also have an influence onhalf-life in blood has been reported (Non Patent Literature 5: D.Bumbaca, AAPSJ, 2012, 14, 3). Moreover, it has been revealed thatantibodies 2,6-sialylated at the nonreducing terminals of theN297-linked sugar chains are active pharmaceutical ingredients in theIVIG (Non Patent Literature 6: Wang, ACS Chem. Biol. 2012, 7, 110-122).Moreover, it is considered that the heterogeneity of N297-linked sugarchains in therapeutic molecules including IgG and Fc fragments has alarge influence on their nature and quality as an active ingredient andthere is an undeniable possibility that contamination with a smallamount of heterogeneously glycosylated molecules significantly changesthe properties of end-products.

In response to this current situation, techniques for making sugarchains homogenous in the production of glycoprotein molecules includingtherapeutic antibodies and antibody Fc regions are being developed.Known methods for making sugar chains to be added to a glycoproteinhomogenous include the transglycosidation reaction using enzymes (NonPatent Literature 6: Wang, ACS Chem. Biol. 2012, 7, 110-122; Non PatentLiterature 7: Wang, Trends Glycosci. Glycotechnol. 2011, 23, 33-52).This is a multi-stage process consisting of cleavage (hydrolysisreaction) of sugar chains and condensation of sugar chains(transglycosylation reaction) in an in vitro environment. In particular,for the purpose of converting N-type sugar chains, a group of enzymesreferred to as endo-β-N-acetylglucosaminidases are used. Such enzymesare required 1) to have an ability to hydrolyze complex-type sugarchains specifically as their substrates and 2) to have an ability tocatalyze the transglycosylation reaction of a particular structure, astheir properties. Endo-β-N-acetylglucosaminidases are isolated fromvarious species and different wild type and mutant enzymes are useddepending on the kind of sugar chains to be modified as substrates.EndoA (enzyme derived from Arthrobacter protophormiae) (Non PatentLiterature 8: Takegawa, Biochem Int. 1991 July; 24(5): 849-55), EndoD(enzyme derived from Streptococcus pneumoniae) (Non Patent Literature 9:Fan, J Biol Chem. 2012 Mar. 30; 287(14): 11272-81), EndoM (enzymederived from Mucor hiemalis) (Non Patent Literature 10: Umekawa, BiochemBiophys Res Commun. 1994 Aug. 30, 203(1): 244-52), EndoH (Non PatentLiterature 11: Robbins, J Biol Chem. 1984 Jun. 25; 259(12): 7577-83),EndoF2 (enzyme derived from Flavobacterium Meningosepticum) (Non PatentLiterature 12: Huang, Chembiochem. 2011 Apr. 11; 12(6): 932-941), EndoE(enzyme derived from Enterococcus faecalis) (Non Patent Literature 13:Collin, JBC 2004, 279-21), EndoS (enzyme derived from Streptococcuspygenes) (Non Patent Literature 14: Collin, EMBO J. 2001 Jun 15; 20(12):3046-55), and the like are used for the purpose of making sugar chainshomogenous, but only EndoS is confirmed to be an enzyme having bothhydrolysis activity and transglycosylation activity on a substrate whichis complex-type N297-linked sugar chains having core fucose (Non PatentLiterature 15: Allhorn, BMC Microbiol. 2008, 8, 3.; Non PatentLiterature 16: Goodfellow, J. Am. Chem. Soc. 2012, 134, 8030-8033).

Further known concerning EndoS is that the hydrolysis activity of EndoSD233Q (in which there is a mutation that substitutes Asp at position233, the nucleophilic catalytic residue among the 2 catalytic residues(which are Asp at position 233 and Glu at position 235) with Gln) issuppressed to some extent. This mutant is known to catalyze thetransglycosylation reaction selectively under conditions in which thereaction system contains a lot of intermediates having sugar chains withoxazolinated reducing terminals (Non Patent Literature 17: Huang, J. Am.Chem. Soc. 2012, 134, 12308-12318; Patent Literature 1: WO 2013/120066A1; Non Patent Literature 18: B. Trastoy et al., PNAS (2014) vol 111,No.18, pp6714-6719). Moreover, many sequences of enzymes classified asbeing EndoS enzymes that are sequences derived from different serotypesof different strains in the species pyogenes have been reported. Inparticular, EndoS2 derived from the serotype M49 (Non Patent Literature19: Sjogren, Biochem J. 2013 Oct 1; 455(1): 107-18; which may also bereferred to as EndoS49) has been reported to have similar substrateproperties.

However, the activity of these known EndoS enzymes and mutant enzymessuch as EndoS D233Q is not sufficient to perform sugar remodeling oftherapeutic antibodies on an industrial scale and a technicalimprovement that makes effective sugar remodeling possible is required.

CITATION LIST Patent Literature

-   Patent Literature 1: WO 2013/120066 A1 pamphlet

Non Patent Literature

-   Non Patent Literature 1: J. N. Arnold et al., Annu. Rev. Immunol,    2007, 25, 21050-   Non Patent Literature 2: R. Jefferis, Biotechnol. Prog., 2005,    21-11-6-   Non Patent Literature 3: Nimmerjahn, Nat. Rev. Immunol. 2008, 8,    34-47-   Non Patent Literature 4: Jefferis Nat. Rev. Drug Discovery 2009, 8,    226-234-   Non Patent Literature 5: D. Bumbaca, AAPSJ, 2012, 14, 3-   Non Patent Literature 6: Wang, ACS Chem. Biol. 2012, 7, 110-122-   Non Patent Literature 7: Wang, Trends Glycosci. Glycotechnol. 2011,    23, 33-52-   Non Patent Literature 8: Takegawa, Biochem Int. 1991 July; 24(5):    849-55-   Non Patent Literature 9: Fan, J Biol Chem. 2012 Mar. 30; 287(14):    11272-81-   Non Patent Literature 10: Umekawa, Biochem Biophys Res Commun. 1994    Aug. 30; 203(1): 244-52-   Non Patent Literature 11: Robbins, J Biol Chem. 1984 Jun. 25;    259(12): 7577-83-   Non Patent Literature 12: Huang, Chembiochem. 2011 Apr. 11; 12(6):    932-941-   Non Patent Literature 13: Collin, JBC 2004, 279-21-   Non Patent Literature 14: Collin, EMBO J. 2001 Jun. 15; 20(12):    3046-55-   Non Patent Literature 15: Allhorn, BMC Microbiol. 2008, 8, 3.;-   Non Patent Literature 16: Goodfellow, J. Am. Chem. Soc. 2012, 134,    8030-8033-   Non Patent Literature 17: Huang, J. Am. Chem. Soc. 2012, 134,    12308-12318-   Non Patent Literature 18: B. Trastoy et al., PNAS (2014) vol 111,    No.18, pp6714-6719-   Non Patent Literature 19: Sjogren, Biochem J. 2013 Oct. 1; 455(1):    107-18

SUMMARY OF INVENTION Technical Problem

An object of the present invention is to provide a novel mutant enzymehaving reduced hydrolysis activity in comparison with EndoS D233Q (whichis known to be a mutant of endo-β-N-acetylglucosaminidase havingspecificity to sugar chains of the Fc regions of IgG and havingsuppressed hydrolysis activity in comparison with EndoS) and maintaininga certain transglycosylation activity.

From a study using the known EndoS D233Q, it was found that since thismutant maintains a certain hydrolysis activity, its transglycosylationrate in the transglycosylation reaction decreases over time after theonset of the reaction. Therefore, it is difficult to acquire an antibodyhaving homogenous sugar chains at high purity by sugar remodeling withEndoS or existing mutants. In particular, in reactions at a large scale,a final-stage antibody separated from the enzyme inevitably contains theantibody with truncated sugar chains since the purification step takes along time.

Furthermore, chemically synthesized oxazoline derivatives of sugarchains used as sugar donors are often expensive and their use in a largequantity leads to an increase in the production cost. Therefore, anenzyme having a reduced hydrolysis activity in comparison with EndoSD233Q and a transglycosylation activity equal to or higher than acertain level is necessary to produce an antibody having homogenoussugar chains at high purity.

Solution to Problem

As a result of diligent studies to achieve the aforementioned object,the present inventors have designed and produced a large number ofmutants with reference to the amino acid sequence information and the 3Dstructure of EndoS D233Q and found that novel mutant enzymes having anadditional mutation(s) of a particular amino acid(s) in addition toD233Q have the desired activity and further studies have been made tocomplete the present invention.

The present invention provides the following inventions:

-   (1) An EndoS mutant enzyme comprising an amino acid sequence of    amino acid numbers 37 to 995 of SEQ ID NO: 2 with an additional    mutation at at least one amino acid position selected from the group    consisting of: amino acid 122 (H122), amino acid 184 (P184), amino    acid 279 (D279), amino acid 282 (Y282), amino acid 303 (Q303), amino    acid 348 (Y348), amino acid 350 (E350), amino acid 402 (Y402), amino    acid 405 (D405), and amino acid 406 (R406), and exhibiting a reduced    hydrolysis activity in comparison with the activity of EndoS D233Q,    the activity being an activity on an N-linked sugar chain    (N297-linked sugar chain) linked to Asn at position 297 in IgG.-   (2) The EndoS mutant enzyme according to (1), wherein the additional    mutation is at 1 to 4 amino acid positions selected from the group    consisting of: H122, P184, D279, Y282, Q303, Y348, E350, Y402, D405,    and R406.-   (3) The EndoS mutant enzyme according to (1), wherein the additional    mutation is a mutation at one amino acid position selected from the    group consisting of: Q303, E350, and D405.-   (4) The EndoS mutant enzyme according to any one of (1) to (3),    wherein the additional mutation is at least one selected from the    following group:-   a mutation at H122 to the amino acids: Gly, Cys, Thr, Ser, Val, Pro,    Ala, Glu, Asp, Leu, Ile, Pro, Met, or Phe;-   a mutation at P184 to the amino acids: Gly, Cys, Thr, Ser, Val, Pro,    Ala, Gln, or Asn;-   a mutation at D279 to the amino acids: Gly, Cys, Thr, Ser, Val, Pro,    Ala, or Gln;-   a mutation at Y282 to the amino acids: Arg, Lys, or His;-   a mutation at Q303 to the amino acids: Met, Pro, or Leu;-   a mutation at Y348 to the amino acids: His or Trp;-   a mutation at E350 to the amino acids: Lys, Arg, His, Tyr, Gln, Asn,    Gly, Cys, Thr, Ser, Val, Pro, or Ala;-   a mutation at Y402 to the amino acids: Phe or Trp;-   a mutation at D405 to the amino acids: Gly, Cys, Thr, Ser, Val, Pro,    or Ala; and-   a mutation at R406 to the amino acids: Gly, Cys, Thr, Ser, Val, Pro,    Ala, Glu, Asp, Gln, or Asn.-   (5) The EndoS mutant enzyme according to (4), wherein the additional    mutation is at least one selected from the following group:-   a mutation at H122 to Ala (H122A) or Phe (H122F);-   a mutation at P184 to Gln (P184Q);-   a mutation at D279 to Ser (D279S) or Gln (D279Q);-   a mutation at Y282 to Arg (Y282R);-   a mutation at Q303 to Leu (Q303L);-   a mutation at Y348 to His (Y348H);-   a mutation at E350 to Ala (E350A), Asn (E350N), Asp (E350D), or Gln    (E350Q);-   a mutation at Y402 to Phe (Y402F) or Trp (Y402W);-   a mutation at D405 to Ala (D405A); and-   a mutation at R406 to Gln (R406Q).-   (6) The EndoS mutant enzyme according to (5), comprising at least    one mutation selected from the group consisting of Q303L, E350A,    E350N, E350D, E350Q, and D405A as the additional mutation.-   (7) The EndoS mutant enzyme according to (6), wherein the additional    mutation is Q303L, E350A, E350N, E350D, E350Q, D405A, H122A/Q303L,    H122F/Q303L, P184Q/Q303L, D279Q/Q303L, D279S/Q303L, Y282R/Q303L,    Q303L/Y348H, Q303L/E350A, Q303L/E350N, Q303L/E350D, Q303L/E350Q,    Q303L/Y402F, Q303L/Y402W, Q303L/D405A, Q303L/R406Q, H122A/E350A,    H122F/E350A, P184Q/E350A, D279Q/E350A, D279S/E350A, Y282R/E350A,    Y348H/E350A, E350A/Y402F, E350A/Y402W, E350A/D405A, E350A/R406Q,    H122A/E350N, H122F/E350N, P184Q/E350N, D279Q/E350N, D279S/E350N,    Y282R/E350N, Y348H/E350N, E350N/Y402F, E350N/Y402W, E350N/D405A,    E350N/R406Q, H122A/E350D, H122F/E350D, P184Q/E350D, D279Q/E350D,    D279S/E350D, Y282R/E350D, Y348H/E350D, E350D/Y402F, E350D/Y402W,    E350D/D405A, E350D/R406Q, H122A/E350Q, H122F/E350Q, P184Q/E350Q,    D279Q/E350Q, D279S/E350Q, Y282R/E350Q, E350Q/Y348H, E350Q/Y402F,    E350Q/Y402W, E350Q/D405A, E350Q/R406Q, H122A/D405A, H122F/D405A,    P184Q/D405A, D279Q/D405A, D279S/D405A, Y282R/D405A, Y348H/D405A, or    D405A/R406Q.-   (8) The EndoS mutant enzyme according to (7), wherein the additional    mutation is Q303L, E350A, E350N, E350D, E350Q, D405A, Q303L/E350A,    Q303L/E350N, Q303L/E350D, Q303L/E350Q, Q303L/D405A, E350A/D405A,    E350N/D405A, E350D/D405A, or E350Q/D405A.-   (9) The EndoS mutant enzyme according to (4), wherein the additional    mutation is H122A, H122F, P184Q, D279Q, D279S, Y282R, Y348H, Y402F,    Y402W, or R406Q.-   (10) The EndoS mutant enzyme according to (1), wherein the activity    on the N297-linked sugar chain keeps a hydrolysis rate of 50% or    less in a hydrolysis reaction in a reaction solution at pH 7.4 after    24 hours later.-   (11) The EndoS mutant enzyme according to (1), wherein the EndoS    mutant enzyme further exhibits an activity on the N297-linked sugar    chain leading to a percent transglycosylation of approximately 60%    or more after 48 hours of a transglycosylation reaction with 5    equivalents of a sugar donor in a reaction solution at pH 7.4.-   (12) A polynucleotide encoding an EndoS mutant enzyme according to    any one of (1) to (11).-   (13) A vector comprising a nucleotide complementary to the    polynucleotide according to (12).-   (14) A host cell transformed with a vector according to (13).-   (15) A method of production of a molecule comprising a    sugar-remodeled Fc region, comprising reacting a molecule comprising    an Fc region of IgG having a core GlcNAc optionally with fucose    addition as an N297-linked sugar chain with a sugar donor having a    structure comprising an oxazolinated GlcNAc in the presence of an    EndoS mutant enzyme according to any one of (1) to (11) to produce a    molecule comprising an Fc region with a N297-linked sugar chain    having a structure in which a sugar chain possessed by the sugar    donor is transferred to the core GlcNAc of the N297-linked sugar    chain.-   (16) The method of production according to (15), wherein the    molecule comprising an Fc region is an IgG monoclonal antibody.

Advantageous Effects of Invention

The novel EndoS mutant enzymes of the present invention have a furtherreduced hydrolysis activity in comparison with EndoS D233Q, which isknown to be a mutant having reduced hydrolysis activity, and maintain atransglycosylation activity equal to or higher than a certain level.Therefore an antibody having homogenous sugar chains can be acquired bysugar remodeling with the mutant enzymes of the present invention at ahigher purity and more efficiently. Moreover, they lead to a decrease inthe production cost of sugar-remodeled antibodies since the amount ofsugar donor used in the sugar remodeling can be decreased.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates a chemical structural formula (left) of SG-Oxa, whichcan be used as a sugar donor in sugar remodeling, and a representation(right) of the sugar chain in symbol form.

FIG. 2 is a schematic view of the hydrolysis reaction of an N297-linkedsugar chain of an antibody using EndoS or an EndoS mutant enzyme.

FIG. 3 schematically illustrates the transglycosylation reaction usingEndoS or an EndoS mutant enzyme.

FIG. 4 illustrates an alignment of the amino acid sequences of EndoSmutant enzymes (EndoS D233Q, EndoS D233Q/Q303L, EndoS D233Q/Q303L/E350Q,EndoS D233Q/Q303L/D405A, EndoS D233Q/E350Q, EndoS D233Q/D405A, and EndoSD233Q/Y402F). The amino acids are numbered based on the full lengthcoding region including the signal sequence (1-36). FIG. 4-1 illustratesan alignment of amino acid numbers 37-176 of the mutant enzymes.

FIG. 4-2 is a continuation of FIG. 4-1 and illustrates an alignment ofamino acid numbers 177-316 of the mutant enzymes.

FIG. 4-3 is a continuation of FIG. 4-2 and illustrates an alignment ofamino acid numbers 317-456 of the mutant enzymes.

FIG. 4-4 is a continuation of FIG. 4-3 and illustrates an alignment ofamino acid numbers 467-596 of the mutant enzymes.

FIG. 4-5 is a continuation of FIG. 4-4 and illustrates an alignment ofamino acid numbers 597-736 of the mutant enzymes.

FIG. 4-6 is a continuation of FIG. 4-5 and illustrates an alignment ofamino acid numbers 737-876 of the mutant enzymes.

FIG. 4-7 is a continuation of FIG. 4-6 and illustrates an alignment ofamino acid numbers 877-995 of the mutant enzymes.

DESCRIPTION OF EMBODIMENTS

The present invention is described in detail below.

The notation of amino acids contained in a molecule herein conforms tothe customs of the field and the position of a mutation is indicated bythe one character notation of the wildtype amino acid (or nucleic acid)and its number (for example, Asp at position 233 is referred to as“D233”). Mutations are indicated by the one character notation of thewildtype amino acid (or nucleic acid), its number, and the one characternotation of the amino acid (or nucleic acid) after the mutation (forexample, the mutation that substitutes Asp at position 233 with Gln isreferred to as “D233Q”). Moreover, particular mutants having a mutationare indicated by the molecule name and the mutation (for example, themutant of EndoS in which Asp at position 233 is substituted with Gln isreferred to as “EndoS D233Q”) and, if the mutant has a plurality ofmutations, the mutations are indicated with a separator “/” between themutations (for example, the mutant of EndoS D233Q having an additionalmutation that substitutes Gln at position 303 with Leu is referred to as“EndoS D233Q/Q303L”).

In the present invention, the term “N297-linked sugar chains” meanssugar chains linked to the Asn side chain at position 297 of an IgGheavy chain. When an IgG is fragmented, a sugar chain linked to thecorresponding Asn in a peptide fragment containing the Asn is alsoincluded within the term “N297-linked sugar chain”. Usually, theN297-linked sugar chains in IgG produced in animals and the like have abasic structure represented by the following formula and Gal or sialicacid may be further added to their nonreducing terminals.

Most N297-linked sugar chains of IgG produced by cells have a variety ofsugar chain structures, including sugar chains in which further sugarchains are linked to a GlcNAc at the reducing terminal(core GlcNAc)and/or moiety at the nonreducing terminals, branched sugars, or the likein this basic structure. The core GlcNAc may be modified into thestructure ((Fucα1,6)GlcNAc) having a core fucose in which fucose is α1,6linked to the 6th position of the core GlcNAc. The branched mannose mayform a tribranched type sugar chain in which a further sugar chaincontaining GlcNAc is linked to the 5th position of mannose. To GlcNAc atthe nonreducing terminals, sugar chains containing Gal or sialic acidmay be linked.

In the present invention, “sugar donors” are sugar chain-containingmolecules having oxazolinated GlcNAc at the reducing terminals of theirsugar chains and molecules with various sugar chain structures areavailable.

When sugar donors are used for sugar remodeling for the purpose of drugdiscovery, it is preferred to employ a sugar donor having a human sugarchain or a human-compatible sugar chain that causes few problems whenapplied to humans. Such sugar chains are sugar chains known to exhibitno antigenicity in the human body and known examples of such sugarchains that are N-linked sugar chains include the high-mannose type, thehybrid type, and the complex type. These 3 have common basic structures.The high-mannose type of sugar chains are those having a mannose-richstructure in which a plurality of mannose residues are connected to the2 branched chains (the 1-3 and 1-6 chains) branched from mannose(β-mannose) at the position close to the reducing terminal. The hybridtype of sugar chains are those in a structure having GlcNAc in one ofthe 2 branched chains (the 1-3 and 1-6 chains) branched from mannose(β-mannose) at the position close to the reducing terminal. The complextype of sugar chains are those that are in a structure having GlcNAc inthe 2 branched chains (the 1-3 and 1-6 chains) branched from mannose(β-mannose) at the position close to the reducing terminal and havevarious structures including those with or without galactose, with orwithout sial, and linkage isomers and regioisomers thereof. (See Table1).

A representative sugar donor is SG-Oxa produced in Example 2 (FIG. 1).

In the present invention, “acceptor molecules” are molecules containinga sugar structure having GlcNAc at a nonreducing terminal and, when theyare reacted with sugar donor molecules in the presence of EndoS or amutant enzyme thereof, they can form the chitobiose structure as aresult of the reaction of the oxazoline rings in the sugar donormolecules with the 4th position of GlcNAc at a nonreducing terminal ofthe acceptor molecules. A typical acceptor molecule is IgG or an Fcfragment thereof that is derived from a monoclonal antibody and has anN297-linked sugar chain only consisting of core GlcNAc optionally linkedwith core fucose. To the core GlcNAc, core fucose may or may not belinked depending on the antibody from which the acceptor molecule isderived or the method of production thereof. Various monoclonalantibodies or molecules comprising an Fc region (such as Fc, CLCH, whichis a combination of CH, only, consisting of the constant region obtainedby deleting the variable region from the heavy chain and CL, only,consisting of the constant region of the light chain) may be used as asource of acceptor molecules, but preferable examples thereof include(Fucα1,6)-GlcNAc-IgG, (Fucα1,6)-GlcNAc-Fc, and (Fucα1,6)-GlcNAc-CLCH andrepresentative examples include (Fucα1,6)-GlcNAc-Trastuzumab, which isproduced in Example 3.

In the present invention, “EndoS” is a kind ofendo-β-N-acetylglucosaminidase (ENGase) derived from Streptococcuspyogenes and the enzyme consists of an amino acid sequence of amino acidnumbers 37 to 995 (amino acids at positions 1 to 36 correspond to asignal sequence) of SEQ ID NO: 1 in which the amino acid at position 233is Asp (EC 3.2.1.96, GH18). EndoS specifically recognizes N297-linkedsugar chains on the Fc site of IgG and has both hydrolysis activity andtransglycosylation activity. The hydrolysis activity of EndoS is theactivity that specifically hydrolyzes the β-1,4-glycosidic bondcontained in the core chitobiose of N297-linked sugar chains having thebasic structure described above (as used herein, unless otherwisespecified, “hydrolysis activity” means this activity; a schematic viewof the reaction is shown in FIG. 2). The transglycosylation activity ofEndoS is the activity that forms a glycosidic bond between the reducingterminal of a sugar chain derived from a sugar donor having oxazolinatedGlcNAc (core fucose may or may not be added) at the reducing terminaland an acceptor molecule containing an Fc site having only core GlcNAcat N297 (this activity is, hereinafter, referred to as“transglycosylation activity”; a schematic view of the reaction is shownin FIG. 3).

The structure of EndoS has been reported to be a 5 domain structure: theendoglycosidase enzymatic domain (catalytic domain: amino acid numbers98 to 445 of SEQ ID NO: 1), the leucine-rich repeat domain (amino acidnumbers 446 to 631 of SEQ ID NO: 1), the hybrid Ig domain (amino acidnumbers 632 to 764 of SEQ ID NO: 1), the carbohydrate-binding module(CBM: amino acid numbers 765 to 923 of SEQ ID NO: 1), and the threehelix-bundle domain (amino acid numbers 924 to 995 of SEQ ID NO: 1) (B.Trastoy et al., PNAS (2014) vol 111, No. 18, pp6714-6719), among whichthe sites that are important to the interaction with antibodies areconsidered to be the following two domains: the catalytic domain andCBM.

EndoS is an enzyme well conserved in Group A Streptococcus (GAS). EndoS2(which may also be referred to as EndoS49; Sjogren, Biochem J. 2013 Oct.1; 455(1): 107-18; US 2014/0302519 A1) found in NZ131 belonging to theserotype M49 of GAS has a sequence identity of 37%, but the importantamino acids composing the catalytic domain are well conserved and EndoS2exhibits a substrate specificity similar to EndoS. As used herein, suchenzymes having an amino acid homology/identity with the active region ofEndoS are referred to as “EndoS-related enzymes”. The important mutationpositions, H122, D233, D279, Q303, E350, Y402, D405, and R406 in EndoSrespectively correspond to H75, D184, D226, Q250, E289, Y339, D342, andR343 in EndoS2. The mutant enzymes of the present invention also includemutant enzymes in which corresponding amino acids are mutated fromrelated enzymes such as EndoS2.

In the present invention, “EndoS D233Q” is a mutant enzyme consisting ofa sequence in which Asp at position 233 in wild type EndoS issubstituted with Gln (the amino acid sequence of amino acid numbers 37to 995 of SEQ ID NO: 1) and the mutant enzyme has the hydrolysisactivity of EndoS suppressed by a certain degree and maintains thetransglycosylation activity at the level equivalent to the wild type.

<Mutant Enzyme of the Present Invention>

The present invention provides EndoS D233Q mutant enzymes that containthe region necessary for the transglycosylation activity in the aminoacid sequence of amino acid numbers 37 to 995 of SEQ ID NO: 2 and thathave an additional mutation at at least one amino acid position selectedfrom the group of positions of certain essential additional mutations,in addition to D233Q; and that have reduced hydrolysis activity toN297-linked sugar chains of IgG in comparison with EndoS D233Q.

In the present invention, the term “additional mutations” meansadditional amino acid alterations of amino acids other than D233 in theamino acid sequence of EndoS D233Q. In the present invention, the “groupof positions of essential additional mutations” is a candidate group ofpositions that are necessarily mutated additionally in the mutantenzymes of the present invention and is the group consisting of thepositions indicated by Xaa in the amino acid sequence of SEQ ID NO: 2,that is to say, H122, P184, D279, Y282, Q303, Y348, E350, Y402, D405,and R406, and is preferably the group consisting of Q303, E350, andD405.

The mutant enzymes of the present invention are characterized by havingboth hydrolysis activity lower than EndoS D233Q and a certain level oftransglycosylation activity (hereinafter, having both activities isreferred to as having “the present enzyme activity”). As to whether amutant enzyme has the present enzyme activity, the hydrolysis andtransglycosylation activities can be evaluated by a method of Example 4described below.

As regards the present enzyme activity that the mutant enzymes of thepresent invention have, the hydrolysis activity is suppressed incomparison with hydrolysis activity of EndoS D233Q and, morespecifically, the percent hydrolysis in conditions at pH 7.4 asdescribed in Example 4 exhibits a value lower than the percenthydrolysis of EndoS D233Q at any one time point from the onset of thereaction to 1 to 48 hours later, but preferably the percent hydrolysisat 24 hours after the onset of the hydrolysis reaction is 50% or less orthe percent hydrolysis at 48 hours later is 60% or less than that ofEndoS D233Q, more preferably the percent hydrolysis is maintained at 40%or less than that of EndoS D233Q for 24 hours from the onset of thehydrolysis reaction, further preferably at 30% or less, even morepreferably at 20% or less for the same time period, and most preferablythe percent hydrolysis exhibited is 0% at all time points and thehydrolysis activity is diminished completely.

As regards the present enzyme activity that the mutant enzymes of thepresent invention have, the transglycosylation activity is at a levelequivalent to the transglycosylation activity of EndoS D233Q and, morespecifically, the percent transglycosylation in conditions at pH 7.4with 5 equivalents of a sugar donor relative to the acceptor molecule asdescribed in Example 4 is equal to or more than the percenttransglycosylation of EndoS D233Q (for example, 70% or more of the valueof EndoS D233Q) at and after a time point of 1 to 48 hours after theonset of the reaction, but preferably the percent transglycosylationachieves 50% or more than that of EndoS D233Q by 48 hours after theonset of the reaction, more preferably the percent transglycosylationachieves 60% or more than that of EndoS D233Q by 48 hours after theonset of the reaction, further preferably the percent transglycosylationachieves 80% or more than that of EndoS D233Q by 48 hours after theonset of the reaction, and even more preferably the percenttransglycosylation achieves 90% or more than that of EndoS D233Q by 48hours after the onset of the reaction.

The mutant enzymes of the present invention do not have to be of thefull length sequence as long as the regions important for thetransglycosylation activity of EndoS D233Q are conserved in the aminoacid sequence of amino acid numbers 37 to 995 of SEQ ID NO: 2. Fromdomain analyses of EndoS, it is known that the catalytic domain (aminoacid numbers 98 to 445 of SEQ ID NO: 2) and CRM (amino acid numbers 765to 923 of SEQ ID NO: 2) are important and mutant enzymes can be used asthe mutant enzymes of the present invention, as long as they containthese domains. In addition, the present invention also includes relatedenzymes such as EndoS2 as long as they contain regions corresponding tothese domain regions and have mutations of amino acids at correspondingpositions (H122, D233, D279, Q303, E350, Y402, D405, and R406 of EndoSrespectively correspond to H75, D184, D226, Q250, E289, Y339, D342, andR343 of EndoS2) as appropriate and display the present enzyme activity.They are preferably polypeptides containing amino acid numbers 98 to 923of SEQ ID NO: 2, more preferably polypeptides containing amino acidnumbers 98 to 995 of SEQ ID NO: 2, and further preferably polypeptidescontaining amino acid numbers 37 to 995 of SEQ ID NO: 2.

In the amino acid sequences of the mutant enzymes of the presentinvention, one to several amino acids may be substituted, deleted,inserted, and/or added at positions other than the positions ofessential mutations described below to the extent that it does notaffect the present enzyme activity. Any positions may be selected forsuch amino acid alterations as long as it does not affect the presentenzyme activity, but the position is preferably a position out of thecatalytic domain (amino acid numbers 98 to 445 of SEQ ID NO: 2) and CRM(amino acid numbers 765 to 923 of SEQ ID NO: 2) and more preferably aposition contained in the regions of amino acid numbers 37 to 97 oramino acid numbers 924 to 995 of SEQ ID NO: 2. In the present invention,the term “several” refers to 20 or less, preferably 10 or less, furtherpreferably 5 or less, and most preferably 4, 3, 2, or 1.

In a mutant enzyme of the present invention, the positions foradditional mutations other than D233Q include at least one of thepositions selected from the group of the positions of the essentialadditional mutations. The number of positions for additional mutationsis not particularly limited as long as the produced mutant enzyme hasthe present enzyme activity, but it is preferably 10 or less, morepreferably 5 or less, and further preferably 4, 3, 2, or 1. When themutant enzyme has additional mutations at plural positions, if there isa mutation at at least one of the group of positions of essentialadditional mutations, then the other positions of mutation are notparticularly limited as long as the produced mutant enzyme has thepresent enzyme activity, but it is preferred that all additionalmutations are in the group of positions of essential additionalmutations. When the mutant enzyme has an additional mutation at aposition other than the positions of essential additional mutations, theregion containing the additional mutation at a position other than thepositions of essential additional mutations is preferably of an aminoacid outside of the catalytic domain (amino acid numbers 98 to 445 ofSEQ ID NO: 2), which relates to the enzyme activity, more preferably theregion of amino acid numbers 37 to 97, 446 to 764, and 924 to 995 of SEQID NO: 2, and further preferably the region of amino acid numbers 37 to97 and 924 to 995 of SEQ ID NO: 2.

In the present invention, the amino acid after performing thesubstitution of the additional mutation is not particularly limited aslong as the finally obtained mutant enzyme has the present enzymeactivity and any of various amino acids such as naturally occurringamino acids, artificially synthesized amino acids, and modified aminoacids thereof may be used, but the amino acid is preferably a naturallyoccurring amino acid, more preferably a naturally occurring L-aminoacid, and further preferably an essential amino acid. Amino acids thatare preferred for the mutant amino acid at one of the positions ofessential additional mutations are illustrated below.

The mutant amino acid at H122 is preferably an amino acid (Gly, Cys,Thr, Ser, Val, Pro, Ala) having a small side chain structure, such asthose that diminish the interaction between the position and thesurrounding amino acids, an amino acid (Glu or Asp) having a minuscharge in the side chain, or an amino acid (Leu, Ile, Pro, Met, Phe)that has no highly reactive functional group in the side chain and morepreferably Ala or Phe.

The mutant amino acid at P184 is preferably an amino acid (Gly, Cys,Thr, Ser, Val, Pro, Ala) having a small side chain structure or an aminoacid (Gln, Asn) that has an amide group in the side chain and morepreferably Gln.

The mutant amino acid at D279 is preferably an amino acid (Gly, Cys,Thr, Ser, Val, Pro, Ala) having a small side chain structure or Gln andmore preferably Ser or Gln.

The mutant amino acid at Y282 is preferably an amino acid (Arg, Lys,His) whose side chain is basic and more preferably Arg.

The mutant amino acid at Q303 is preferably a hydrophobic amino acid(Met, Pro, Leu) and more preferably Leu.

The mutant amino acid at Y348 is preferably an amino acid (His, Trp)having a ring structure in the side chain and more preferably His.

The mutant amino acid at E350 is preferably an amino acid (Lys, Arg,His, Tyr, Gln, Asn) having a large side chain capable of forming ahydrogen bond or an amino acid (Gly, Cys, Thr, Ser, Val, Pro, Ala)having a small side chain structure, such as those that diminish theinteraction between the position and the surrounding amino acids, andmore preferably Ala, Asn, Asp, or Gln.

The mutant amino acid at Y402 is preferably a large amino acid having anaromatic ring in the side chain, Phe or Trp.

The mutant amino acid at D405 is preferably an amino acid (Gly, Cys,Thr, Ser, Val, Pro, Ala) having a small side chain structure, such asthose that diminish the interaction between the position and thesurrounding amino acids and more preferably Ala.

The mutant amino acid at R406 is preferably an amino acid (Gly, Cys,Thr, Ser, Val, Pro, Ala) having a small side chain structure, such asthose that diminish the interaction between the position and thesurrounding amino acids, or an amino acid (Glu, Asp) having a minuscharge in the side chain, or an amino acid (Gln, Asn) that has an amidegroup in the side chain and more preferably Ala or Gln.

The mutation at the position of an essential additional mutation asdescribed above may be alone or combined with a mutation at the positionof another essential additional mutation and/or a mutation at anotherposition. The combination of mutations at positions of essentialadditional mutations may be any combination, but is preferably acombination containing a mutation at at least Q303, E350, or D405 andmore preferably a combination containing at least Q303L, E350A, E350N,E350Q, or D405A.

Examples of the combination of additional mutations containing Q303L caninclude H122A/Q303L, H122F/Q303L, P184Q/Q303L, D279Q/Q303L, D279S/Q303L,Y282R/Q303L, Q303L/Y348H, Q303L/E350A, Q303L/E350N, Q303L/E350D,Q303L/E350Q, Q303L/Y402F, Q303L/Y402W, Q303L/D405A, and Q303L/R406Q.

Examples of the combination of additional mutations containing E350A caninclude H122A/E350A, H122F/E350A, P184Q/E350A, D279Q/E350A, D279S/E350A,Y282R/E350A, Y348H/E350A, E350A/Y402F, E350A/Y402W, E350A/D405A, andE350A/R406Q.

Examples of the combination of additional mutations containing E350N caninclude H122A/E350N, H122F/E350N, P184Q/E350N, D279Q/E350N, D279S/E350N,Y282R/E350N, Y348H/E350N, E350N/Y402F, E350N/Y402W, E350N/D405A, andE350N/R406Q.

Examples of the combination of additional mutations containing E350D caninclude H122A/E350D, H122F/E350D, P184Q/E350D, D279Q/E350D, D279S/E350D,Y282R/E350D, Y348H/E350D, E350D/Y402F, E350D/Y402W, E350D/D405A, andE350D/R406Q.

Examples of the combination of additional mutations containing E350Q caninclude H122A/E350Q, H122F/E350Q, P184Q/E350Q, D279Q/E350Q, D279S/E350Q,Y282R/E350Q, Y348H/E350Q, E350Q/Y402F, E350Q/Y402W, E350Q/D405A, andE350Q/R406Q.

Examples of the combination of additional mutations containing D405A caninclude H122A/D405A, H122F/D405A,

P184Q/D405A, D279Q/D405A, D279S/D405A, Y282R/D405A, Y348H/D405A,Y402F/D405A, Y402W/D405A, or D405A/R406Q.

Preferred mutant enzymes of the present invention are EndoS D233Q/Q303L,EndoS D233Q/E350A, EndoS D233Q/E350N, EndoS D233Q/E350D, EndoSD233Q/E350Q, EndoS D233Q/D405A, EndoS D233Q/Q303L/E350A, EndoSD233Q/Q303L/E350N, EndoS D233Q/Q303L/E350D, EndoS D233Q/Q303L/E350Q,EndoS D233Q/Q303L/D405A, EndoS D233Q/E350A/D405A, EndoSD233Q/E350N/D405A, EndoS D233Q/E350D/D405A, EndoS D233Q/E350Q/D405A, orEndoS D233Q/Y402F, more preferably EndoS D233Q/Q303L, EndoSD233Q/Q303L/E350Q, EndoS D233Q/Q303L/D405A, EndoS D233Q/E350A, or EndoSD233Q/Y402F.

<Gene, Host Cell, Method of Producing Enzyme>

The present invention further provides a recombinant gene encoding amutant enzyme having an additional mutation in EndoS D233Q as describedabove, a gene construct such as a plasmid or an expression vectorcomprising the recombinant gene, a host cell transformed with the geneconstruct, a method of producing the mutant enzyme of the presentinvention, comprising the step of collecting the mutant enzyme of thepresent invention from a culture of the host cell, and the like. Therecombinant gene, gene construct, host cell, and the like can be madeaccording to known genetic engineering techniques based on the aminoacid sequences of the mutant enzymes of the present invention.

With regard to a recombinant gene encoding a mutant enzyme of thepresent invention, a polynucleotide such as a cDNA encoding an aminoacid sequence containing an additional mutation of interest can be madebased on the nucleotide sequence of EndoS D233Q set forth in nucleotidenumbers 109 to 2985 (1 to 108 is the region encoding the signal peptide)of SEQ ID NO: 3. For example, the nucleotide sequence encoding EndoSD233Q/Q303L is a nucleotide sequence modified from the nucleotidesequence of nucleotide numbers 109 to 2985 of SEQ ID NO: 3 bysubstituting the nucleotides CAG at position 907 to 909 with CTG.Moreover, the nucleotide sequence encoding EndoS D233Q/E350A (N, Q) is anucleotide sequence modified from the nucleotide sequence of nucleotidenumbers 109 to 2985 of SEQ ID NO: 3 by substituting the nucleotides GAAat position 1048 to 1050 with GCA (AAT in E350N and CAG in E350Q).Moreover, the nucleotide sequence encoding EndoS D233Q/D405A is anucleotide sequence modified from the nucleotide sequence of nucleotidenumbers 109 to 2985 of SEQ ID NO: 3 by substituting the nucleotides GATat position 1213 to 1215 with GCA.

Host cells (cells, such as animal cells, plant cells, Escherichia coli,yeast, or the like, usually used for protein production may be selectedas appropriate) transformed by the introduction of a gene encoding amutant enzyme of the present invention are cultured under appropriateconditions depending on the kind of cell and the mutant enzyme of thepresent invention can be collected from the culture. The collection ofthe mutant enzyme is performed by combining usual purificationtechniques based on the physical properties of the enzyme asappropriate. To facilitate the collection, the gene construct may bedesigned to express the mutant enzyme in a form with a tag peptide suchas GST connected to the mutant enzyme in advance to make collectionusing affinity to the tag peptide possible. The tag peptide may beremoved after purification, but, when it has no effect on the enzymeactivity, a mutant enzyme with the tag peptide connected thereto may beused for reactions such as sugar remodeling. The mutant enzymes of thepresent invention include such enzymes having an amino acid sequencecomprising that of a tag peptide connected thereto.

<Sugar Remodeling>

The present invention provides a method of sugar remodeling of anN297-linked sugar chain on IgG or a molecule comprising an Fc regionusing an EndoS mutant enzyme of the present invention and IgG or amolecule comprising an Fc region having an N297-linked sugar chainconsisting of a substantially homogenous structure obtained by the sugarremodeling.

The term “sugar remodeling” means a method for producing IgG or amolecule comprising an Fc region having N297-linked sugar chains whichhave a homogenous sugar chain structure derived from a sugar donor byfirst producing acceptor molecules in which N297-linked sugar chains ofthe molecule comprising an Fc region, such as IgG or a Fc fragment orCLCH (which only consists of the constant region) of IgG of a particularmonoclonal antibody, are cut off except for a core GlcNAc (a core fucosemay be added thereto); and then transferring a sugar chain derived froma sugar donor onto the core GlcNAc of the acceptor molecule using thetransglycosylation activity of an EndoS mutant enzyme of the presentinvention.

The IgG, or molecule comprising an Fc region used in sugar remodeling,may be one derived from an IgG heavy chain consisting of the same aminoacid sequence and produced in a form having an N297-linked sugar chainand methods of production thereof are not limited, but IgG produced by acommonly known method of producing a monoclonal antibody, CLCH of theIgG, or an Fc fragment obtained by enzymatic treatment thereof may alsobe used. In addition, a mixture of samples obtained by different methodsof production in different lots may be used as such IgG or Fc fragment.

A method for preparing an acceptor molecule to be used in sugarremodeling may comprise preparing the acceptor molecule by treating theIgG or molecule comprising an Fc region described above with an ENGasewhich maintains the activity of specifically hydrolyzing the1,4-glycosidic bond between GlcNAc in the core chitobiose structure ofthe N297-linked sugar chain. In this case, as the ENGase, variousenzymes including EndoA, EndoD, EndoE, and EndoS may be used, but theENGase is preferably wild type EndoS.

As the sugar donor to be used in sugar remodeling, molecules havingvarious sugar chain structures may be employed. However, for the purposeof using the antibody after remodeling as an antibody medicine, it ispreferred to employ a sugar donor having a human sugar chain structure,which is similar to, or the same as, the sugar chain structure thathumans have or a human-compatible sugar chain structure. Representativeexamples of such a sugar donor can include molecules having a structuremodified from the aforementioned basic structure of N-linked sugarchains by removing the core GlcNAc and oxazolinating the second GlcNAcfrom the reducing terminal and the sugar donor is preferably SG-Oxaconsisting of the structure illustrated in FIG. 1.

As the reaction conditions for the hydrolysis reaction in sugarremodeling, those in commonly known methods for EndoS may be employed.The reaction is performed in a buffer solution, which may be selected asappropriate from buffer solutions used in usual enzymatic reactions:such as a citrate buffer solution (pH 3.5-5.5), an acetate buffersolution (pH 4.5-6.0), a phosphate buffer solution (pH 6.0-7.5), aMOPS-NaOH buffer solution (pH 6.5-8.0), and a Tris-HCl buffer solution(pH 7.0-9.0). A preferred buffer solution is a Tris-HCl buffer solution(pH 7.0-9.0). To the reaction solution, an additive that does notinhibit the enzymatic reaction may be added for the purpose ofstabilizing the enzyme, but it is not necessary.

The reaction temperature may be selected in the range from 10° C. to 50°C. as appropriate, but the preferable temperature is from 25° C. to 38°C.

The reaction time may be selected as appropriate in the range from 10minutes to 96 hours, but the end of the reaction may be determined bycollecting small quantities of the reaction solution over time andconfirming the degree of progress of hydrolysis. In general, the degreeof progress of the sugar chain hydrolysis reaction may be monitored bysodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE),fully automatic electrophoretic systems, liquid chromatography-massspectrometry (LC-MS), or the like. In this patent, a commerciallyavailable antibody or a sugar remodeled antibody was fragmented intoheavy and light chains and then they were examined with a fullyautomatic electrophoretic system to confirm that only the heavy chain,to which N297-linked sugar chains were added, was changed in theretention time.

The reaction conditions for the transglycosylation in sugar remodelingmay be selected as appropriate based on conditions known for otherenzymes.

The reaction is performed in a buffer solution and such a buffer ispreferably one that does not promote the degradation of SG-Oxa and maybe selected from a phosphate buffer solution (pH 6.0-7.5), a MOPS-NaOHbuffer solution (pH 6.5-8.0), and a Tris-HCl buffer solution (pH7.0-9.0) as appropriate. A preferred buffer is a Tris-HCl buffersolution (pH 7.0-9.0). Into the reaction solution, an additive that doesnot inhibit the enzymatic reaction may be added for the purpose ofstabilizing the enzyme, but it is not necessary.

The reaction temperature may be selected in the range from 10° C. to 50°C. as appropriate, but the preferable temperature is from 25° C. to 38°C.

The reaction time may be selected as appropriate in the range from 10minutes to 96 hours, but the end of the reaction may be determined bycollecting small quantities of the reaction solution over time andconfirming the degree of progress of the transglycosylation reaction. Ingeneral, the degree of progress of transglycosylation reaction may bemonitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE), fully automatic electrophoretic systems, liquidchromatography-mass spectrometry (LC-MS), or the like. In this patent, acommercially available antibody or a sugar remodeled antibody wasfragmented into heavy and light chains and then they were examined witha fully automatic electrophoretic system to confirm that only the heavychain, to which N297-linked sugar chains were added, was changed in theretention time.

EXAMPLES

The present invention is specifically described referring to theExamples below. The description in the Examples is an example ofembodiments of the present invention and the present invention is notlimited thereto.

Example 1 is a preparation example of EndoS mutant enzymes used in thepresent invention. Example 2 is a production example of the sugar donorused in the present invention. Example 3 is a preparation example of theacceptor molecule used in measuring transglycosylation activity. Example4 is a measurement example of the percent hydrolysis by the EndoS mutantenzymes of the present invention and a measurement example of thepercent transglycosylation by the EndoS mutant enzymes of the presentinvention.

The protein concentrations described herein were quantified with anultramicrospectrophotometer NanoDrop 1000 (a product made by ThermoFisher Scientific) or NanoDrop 2000 (a product made by Thermo FisherScientific).

The mass of sugar remodeled antibodies ((Fucα1,6)GlcNAc-Trastuzumab andSG-Trastuzumab) was confirmed by the following method. The sugarremodeled antibodies were fragmented into heavy and light chains andthen their peaks were separated with an analytical column and analyzedby mass spectrometry. Apparatuses used were ACUITY UPLC (a product madeby Waters), SYNAPT G2-S (Waters), and BEH Phenyl column (1.7 μm, 1.0×50mm), the mobile phase used was acetonitrile with 0.05% trifluoroaceticacid in a gradient changing from 25% to 35% in 3 minutes, and theanalysis was conducted at a flow rate of 0.34 μL/min at 80° C.

Example 1 Preparation of Reaction Solution of EndoS Mutant Enzymes (1-1)Design of EndoS Mutant Enzymes

EndoS mutants that were considered to achieve suppressed hydrolysisactivity while maintaining the transglycosylation activity of EndoSD233Q by introducing a mutation(s) into EndoS D233Q were designed. Basedon the 3D structure information (PDB ID:4NUY) of EndoS, the catalyticdomain of EndoS was divided into 3 groups of sites: sites predicted tobe involved in the recognition of sugar chains, sites in the vicinity ofthe active center, and sites predicted to be involved in the recognitionof antibodies. Mutations were introduced to the sites. The 6 residuesH122, Y348, E350, Y402, D405 and R406 were selected as the sitespredicted to be involved in the recognition of sugar chains and enzymeswere designed such that the interaction formed by the amino acidresidues described above should be cut or such that amino acid residueshaving a large side chain among the selected amino acids should besubstituted with amino acid residues having a small side chain in orderto make turnover of coupling and dissociation of the sugar chainefficient. P184, D279, and Q303 were selected as sites in the vicinityof the active center and mutations were designed to substitute them witha wide variety of amino acids including those having properties similarto the wildtype amino acid residue or those having properties differentfrom the wildtype amino acid residue. Y282 is selected as one of thesites predicted to be involved in the recognition of antibodies and amutation was designed to substitute the amino acid residue with a basicamino acid in order to strengthen the interaction with the minus chargenear Asn 297 to which sugar chains of antibodies link. The mutantenzymes set forth in Table 2 were designed in the 3 sites describedabove or in consideration of combined mutations among the 3 sites.

(1-2) Expression of EndoS Mutant Enzymes

Escherichia coli was transformed by adding 3 μL of a plasmid (pGEX4T3,50 μg/μL) for protein expression containing a gene encoding the aminoacid sequence of each of the EndoS mutant enzymes designed based on(1-1) above to 50 μL of ice-cooled Escherichia coli BL21 (DE3) strainhaving a competency of 10⁷ cfu/μL and heating the mixture at 37° C. for40 seconds. These Escherichia coli cells were cultured overnight on LBagar medium containing 100 μg/mL of ampicillin and colonies obtained onthe next day were picked up and cultured with shaking in 1 L of TBmedium containing 100 μg/mL of ampicillin at 37° C. until O.D. 600reached 0.8. After O.D. 600 increased, the culture temperature waslowered to 16° C. and 1 hour later isopropyl β-D-1-thiogalactopyranoside(IPTG) was added to a final concentration of 0.1 mM to induce theexpression of the EndoS mutant enzyme overnight. On the next day, cellswere collected by centrifuging the liquid culture at 5000×G for 10minutes and 50 mL of PBS buffer with 40 U/mL DNase I and 0.5 mg/mLLysozyme was added to resuspend the cells. The cells were disrupted bysonicating the obtained bacterial suspension and centrifuged at 20000×gfor 30 minutes to obtain the target EndoS mutant enzyme in the solublefraction.

(1-3) Purification of EndoS Mutant Enzymes

The soluble fractions of the EndoS mutant enzymes obtained in (1-2)above were filtrated through a PVDF membrane having a pore size of 0.45μm and the solutions after the filtration were purified by the 2 stepsprocess by glutathione affinity chromatography and gel filtrationchromatography.

First, the total amounts of the soluble fractions of the EndoS mutantenzymes filtrated through the PVDF membrane were each applied to aglutathione sepharose 4B column equilibrated with PBS and the column waswashed with 3 or more column volumes of PBS buffer. Subsequently, theGST-fused EndoS mutant enzyme was eluted with PBS buffer containing 10mM glutathione in the reduced form and concentrated by ultrafiltration(Amicon Ultra-15 30K). After the concentration, the concentrate wasseparated on HiLoad 26/60 Superdex 200 pg column (GE HealthcareBioscience) equilibrated with PBS to remove glutathione used for theelution and obtain the final purified sample. The yields of the EndoSmutant enzymes prepared in this way are shown in Table 2.

The obtained EndoS mutant enzyme solutions were prepared at 2 mg/mL andthese were used for the measurement of hydrolysis and transglycosylationactivities.

TABLE 2 Yield of EndoS mutant enzymes Concen- Concen- tration Vol-tration Vol- (mg/ ume (mg/ ume Mutant mL) (mL) Mutant mL) (mL) D233Q 3.31.1 D233Q/D405A 7 0.13 H122A/D233Q 5 0.1 D233Q/R406A 5.6 0.9 H122F/D233Q7.8 0.07 D233Q/R406Q 4 0.5 P184Q/D233Q 3.3 0.9 D233Q/D279Q/Y282R 3.3 0.4D233Q/D279S 6.4 0.18 D233Q/Y282R/D405A 2.5 0.4 D233Q/D279Q 3 2.8D233Q/Q303L/Y348H 2.8 1.1 D233Q/Y282R 4.3 0.5 D233Q/Q303L/E350A 2.1 1.3D233Q/Q303L 4.3 2.6 D233Q/Q303L/E350Q 4 0.8 D233Q/Y348H 3.7 2.6D233Q/Q303L/E350D 5.4 0.1 D233Q/E350A 3.8 0.8 D233Q/Q303L/Y402F 2.9 1.2D233Q/E350N 2.7 0.8 D233Q/Q303L/D405A 5.9 0.5 D233Q/E350Q 4.6 2.6D233Q/Y348H/Y402F 3.9 0.85 D233Q/E350D 4.2 2.6 D233Q/E360A/Y402F 3.9 0.8D233Q/Y402F 4.4 2.8 D233Q/E350A/R406A 2.3 1 D233Q/Y402W 3.9 0.7D233Q/Y402F/D405A 3.3 1.05

Example 2 Preparation of Reaction Solution of SG-Oxa (Compound ofStructure in FIG. 1)

SG-Oxa used as a sugar donor in the following Examples was produced bythe following process.

An aqueous solution (520 μl) of2-chloro-1,3-dimethyl-1H-benzimidazol-3-ium chloride (CDMBI) (a productmade by FUSHIMI Pharmaceutical Co., Ltd.) (53.7 mg, 245 μmol) was addedto disialooctasaccharide (Tokyo Chemical Industry Co., Ltd., 100 mg,49.5 μmol). To the reaction solution after cooling on ice, an aqueoussolution (520 μl) of tripotassium phosphate (158 mg, 743 μmol) was addedand the mixture was stirred on ice for 2 hours. The obtained reactionsolution was ultrafiltered with Amicon Ultra (Ultracel 30K, a productmade by Merck Millipore) to remove solid materials. The filtrate waspurified by gel filtration chromatography. The apparatus used wasPurif-Rp2 (a product made by Shoko Scientific Co., Ltd.), the columnused was HiPrep 26/10 Desalting (a product made by GE Healthcare), themobile phase used was a 0.03% —NH3 aqueous solution, the flow rate was10 ml/min, and the fraction volume was 10 ml. The fractions containingthe target product according to UV detection (220 nm) during elutionwere pooled together, a 0.1 N aqueous solution of sodium hydroxide (100μl) was added to the pool, and the mixture was freeze-dried to obtainthe target SG-Oxa as a colorless solid (87.0 mg, 43.4 μmol, 88% yield).

From the NMR chart of the obtained compound, it was confirmed to be thetarget compound (HELVETICA CHIMICA ACTA, 2012, 95, 1928-1936).

To the obtained SG-Oxa (1.19 mg) 50 mM tris buffer solution (pH 7.4)(23.8 μl) was added to prepare 50 mg/ml SG-Oxa solution (50 mM trisbuffer solution pH 7.4). This was used for the measurement oftransglycosylation activity.

Example 3 Preparation of (Fucα1,6)GlcNAc-Trastuzumab

(Fucα1,6)GlcNAc-Trastuzumab to be used as an acceptor molecule in thetransglycosylation reaction was produced by the following process.

A 1.18 mg/ml wild type EndoS solution (PBS) (10 μl) was added to an 11.3mg/ml Trastuzumab (a product made by Genentech) solution (50 mM trisbuffer solution pH 8.0) (2 ml) and the mixture was incubated at 30° C.for 5 hours. The progress of the reaction was confirmed using theExperion electrophoresis station. After the end of the reaction,purification by affinity chromatography and purification with ahydroxyapatite column were performed.

(1) Purification with affinity column

-   Purification apparatus: AKTA avant 25 (a product made by GE    Healthcare)-   Column: HiTrap Protein A HP column (5 ml) (a product made by GE    Healthcare)-   Flow rate: 5 ml/min (1 ml/min at the time of charge)

At the time of binding to the column, the reaction solution obtained asdescribed above was added onto the top of the column, 1 CV of thebinding buffer (20 mM phosphate buffer solution (pH 7.0)) was run at 1ml/min, and further 5 CV was run at 5 ml/min. At the time of theintermediate washing, 15 CV of the washing solution (20 mM phosphatebuffer solution (pH 7.0), 0.5 M sodium chloride solution) was run. Atthe time of elution, 6 CV of the elution buffer (ImmunoPure IgG Eutionbuffer, a product made by PIERCE) was run. The eluate was immediatelyneutralized with 1 M tris buffer solution (pH 9.0). The fractionsdetected by UV detection (280 nm) during elution were examined with theultramicrospectrophotometer NanoDrop 1000 (a product made by ThermoFisher Scientific) and the Experion electrophoresis station (a productmade by BIO-RAD).

The fractions containing the target were concentrated with Amicon Ultra(Ultracel 30K, a product made by Merck Millipore) and buffer (5 mMphosphate buffer solution, 50 mM 2-morpholinoethanesulfonic acid (MES)solution, pH 6.8) exchange was conducted.

(2) Purification with hydroxyapatite column

-   Purification apparatus: AKTA avant25 (a product made by GE    Healthcare)-   Column: Bio-Scale Mini CHT Type I cartridge (5 ml) (a product made    by BIO-RAD)-   Flow rate: 5 ml/min (1 ml/min at the time of charge)

The solution obtained in (1) above was added onto the top of the columnand 4 CV of Solution A (5 mM phosphate buffer solution, 50 mM2-morpholinoethanesulfonic acid (MES), pH 6.8) was run. Then, elutionwas conducted using Solution A and Solution B (5 mM phosphate buffersolution, 50 mM 2-morpholinoethanesulfonic acid (MES), pH 6.8, 2 Msodium chloride solution). The elution conditions were SolutionA:Solution B=100:0 to 0:100 (15 CV).

The fractions detected by UV detection (280 nm) during elution wereexamined with the ultramicrospectrophotometer NanoDrop 1000 (a productmade by Thermo Fisher Scientific) and the Experion (™) electrophoresisstation (BIO-RAD).

The fractions containing the target were concentrated using Amicon Ultra(Ultracel 30K, a product made by Merck Millipore) and buffer exchange to50 mM phosphate buffer solution (pH 6.0) was conducted to obtain a 9.83mg/ml (Fucα1,6)GlcNAc-Trastuzumab solution (phosphate buffer solution,pH 6.0) (1.8 ml). LC-MS:

-   calculated for the heavy chain of (Fuca', 6) GlcNAc-Trastuzumab,    M=49497.86 found (m/z), 49497 (deconvolution data).-   calculated for the light chain of (Fuca', 6) GlcNAc-Trastuzumab,    M=23439.1, found (m/z), 23439.1 (deconvolution data).

Example 4 Measurement of Hydrolysis and Transglycosylation Activities ofEndoS Mutant Enzymes (pH 7.4) (4-1) Measurement of Hydrolysis Activity

The hydrolysis activity of the EndoS mutant enzymes on N297-linked sugarchains of commercially available Trastuzumab was measured as follows. Aschematic view of the hydrolysis reaction is illustrated in FIG. 2.

A 20 mg/ml Trastuzumab solution to be used as a substrate solution forthe hydrolysis reaction was prepared as follows. Otsuka distilled water(10 ml) was added to 100 mM tris buffer solution (pH7.4, a product madeby CALBIOCHEM) (10 ml) to prepare 50 mM tris buffer solution (pH 7.4)(40 ml). To commercially available Trastuzumab (440 mg/vial, a productmade by Genentech), the solubilization liquid (20 ml) attached theretowas added to prepare a Trastuzumab (ca. 21 mg/ml) solution.Ultrafiltration of the Trastuzumab (ca. 21 mg/ml) solution (2 ml) withAmicon Ultra (Ultracel 30K, a product made by Merck Millipore) wasperformed to displace the solvent with 50 mM tris buffer solution pH 7.4prepared as described above and obtain the 20 mg/ml Trastuzumab solution(50 mM tris buffer solution pH 7.4).

To the substrate solution (25 μl) prepared as described above, the 2.0mg/ml EndoS D233Q solution (5 μl) prepared in Example 1 was added andthe mixture was incubated at 30° C. for 48 hours. At 1, 2, 4, 8, 24, and48 hours after the onset of the reaction, a portion of this reactionsolution was collected and the degree of progress of the reaction wasmeasured using an Experion (™) electrophoresis station (BIO-RAD). Forthe measurement, measurement samples were prepared according to theprotocol attached to the apparatus. In this process, the collectedreaction solution is exposed to a solution containing dithiothreitol andheated at 95° C. for 5 minutes and the hydrolysis reaction is stoppedimmediately.

The obtained measurement samples were transferred to Experion (™) Pro260Chips and measured according to the protocol attached to an Experion (™)electrophoresis station (BIO-RAD). From the obtained chromatogram, theunreacted substrate and the hydrolyzed product were confirmed asseparated peaks. From the ratio of the peak areas of the unreactedsubstrate and the hydrolyzed product, the percent hydrolysis wascalculated by the following calculation formula.

Percent hydrolysis (%)=[Peak area of H chain derived from(Fucα1,6)GlcNAc-Trastuzumab]/{[Peak area of H chain derived fromcommercially available Trastuzumab]+[Peak area of H chain derived from(Fucα1,6)GlcNAc-Trastuzumab]}×100

Similarly, the percent hydrolysis of the other EndoS mutant enzymesprepared in Example 1 at each reaction time was calculated (Table 3).

(4-2) Measurement of Transglycosylation Activity

The transglycosylation activity of the EndoS mutant enzymes produced inExample 1 was measured by the following method. SG-Oxa prepared inExample 2 and (Fucα1,6)GlcNAc-Trastuzumab prepared in Example 3 wereused as the sugar donor and the acceptor molecule, respectively. Aschematic view of transglycosylation reaction is illustrated in FIG. 3.

From the 9.83 mg/ml (Fucα1,6)GlcNAc-Trastuzumab solution (phosphatebuffer solution, pH 6.0) prepared in Example 3, a 20 mg/ml(Fucα1,6)GlcNAc-Trastuzumab solution (50 mM tris buffer solution pH 7.4)was prepared according to a method similar to the preparation of thesubstrate solution as described in (4-1). To the 20 mg/ml(Fucα1,6)GlcNAc-Trastuzumab solution (50 mM tris buffer solution pH7.4), the 50 mg/ml SG-Oxa solution (50 mM tris buffer solution pH 7.4)(1.07 μl) prepared in Example 2 and the 2.0 mg/ml EndoS D233Q solution(8 μl) prepared in Example 1 were added and the mixture was incubated at30° C. for 48 hours. At the time points of 1, 2, 4, 8, 24, and 48 hoursafter the onset of the reaction, a portion of this reaction solution wascollected and the degree of progress of the reaction was measured usingan Experion (™) electrophoresis station. For the measurement,measurement samples were prepared according to the protocol attached tothe apparatus. In this process, the collected reaction solution isexposed to a solution containing dithiothreitol and heated at 95° C. for5 minutes and the transglycosylation reaction is stopped immediately.

The obtained measurement samples were transferred to Experion (™) Pro260Chips and measured according to the protocol attached to an Experion (™)electrophoresis station (BIO-RAD). From the obtained chromatogram, theunreacted substrate and the transglycosylation product were confirmed asseparated peaks. From the ratio of the peak areas of the unreactedsubstrate and the transglycosylation product, the percenttransglycosylation was calculated by the following calculation formula.

Percent transglycosylation (%)=[Peak area of H chain derived fromSG-Trastuzumab]/{[Peak area of H chain derived from(Fucα1,6)GlcNAc-Trastuzumab]+[Peak area of H chain derived fromSG-Trastuzumab]}×100

Similarly, the percent transglycosylation of the other EndoS mutantenzymes prepared in Example 1 at each reaction time was calculated(Table 3).

SG-Trastuzumab, which is a transglycosylation product, was purified by amethod similar to the purification of (FucU1,6)GlcNAc-Trastuzumab inExample 3. The obtained LC-MS analysis data of SG-Trastuzumab are shownbelow.

calculated for the heavy chain of SG-Trastuzumab,

-   M=51500.6 Da; found (m/z), 51501 (deconvolution data).-   calculated for the light chain of SG-Trastuzumab,-   M=23439.1 Da, found (m/z), 23439 (deconvolution data).

TABLE 3 Change over time of percent hydrolysis and percenttransglycosylation of EndoS mutant enzymes (pH 7.4) Percent hydrolysis(%) Percent transglycosylation (%) Mutant 1 h 2 h 4 h 8 h 24 h 48 h 1 h2 h 4 h 8 h 24 h 48 h D233Q 0 11 17 30 55 71 52 83 82 80 75 66H122A/D233Q 0 0 21 26 45 54 76 84 85 81 78 71 H122F/D233Q 0 0 0 0 0 0 38 13 28 52 60 D233Q/D279S 0 0 0 0 0 19 21 35 50 64 66 66 D233Q/D279Q 0 00 7 22 30 47 63 77 78 78 74 D233Q/Q303L 0 0 12 20 34 42 33 51 79 94 9691 D233Q/Y348H 0 0 16 22 35 48 53 63 72 75 74 73 D233Q/E350A 0 0 0 12 2832 48 69 86 91 89 87 D233Q/E350N 0 0 0 13 27 37 52 77 89 91 85 84D233Q/E350Q 0 18 24 28 45 53 75 92 91 90 82 74 D233Q/E350D 0 0 0 12 2532 49 66 82 89 89 88 D233Q/Y402F 0 0 0 0 0 15 36 49 66 77 82 82D233Q/D405A 0 0 11 19 34 48 78 90 92 92 85 83 D233Q/R406Q 0 0 0 0 0 0 814 23 33 48 53 D233Q/Y282R/D405A 0 0 0 0 0 0 16 25 41 56 66 71D233Q/Q303L/E350A 0 0 0 0 0 0 5 9 13 24 42 49 D233Q/Q303L/E350Q 0 0 0 00 12 15 33 50 69 87 92 D233Q/Q303L/E350D 0 0 0 0 0 0 5 12 21 34 58 66D233Q/Q303L/Y402F 0 0 0 0 0 0 5 9 15 27 44 52 D233Q/Q303L/D405A 0 0 0 00 0 10 19 30 43 63 72 D233Q/Y402F/D405A 0 0 0 0 0 0 12 22 39 55 72 77

1. An EndoS mutant enzyme comprising an amino acid sequence of aminoacid numbers 37 to 995 of SEQ ID NO: 2 with an additional mutation at atleast one amino acid position selected from the group consisting of:amino acid 122 (H122), amino acid 184 (P184), amino acid 279 (D279),amino acid 282 (Y282), amino acid 303 (Q303), amino acid 348 (Y348),amino acid 350 (E350), amino acid 402 (Y402), amino acid 405 (D405), andamino acid 406 (R406), and exhibiting a reduced hydrolysis activity incomparison with the activity of EndoS D233Q, the activity being anactivity on an N-linked sugar chain (N297-linked sugar chain) linked toAsn at position 297 in IgG.
 2. The EndoS mutant enzyme according toclaim 1, wherein the additional mutation is at 1 to 4 amino acidpositions selected from the group consisting of: H122, P184, D279, Y282,Q303, Y348, E350, Y402, D405, and R406.
 3. The EndoS mutant enzymeaccording to claim 1, wherein the additional mutation is a mutation atone amino acid position selected from the group consisting of: Q303,E350, and D405.
 4. The EndoS mutant enzyme according to any one ofclaims 1 to 3, wherein the additional mutation is at least one selectedfrom the following group: a mutation at H122 to the amino acids: Gly,Cys, Thr, Ser, Val, Pro, Ala, Glu, Asp, Leu, Ile, Pro, Met, or Phe; amutation at P184 to the amino acids: Gly, Cys, Thr, Ser, Val, Pro, Ala,Gln, or Asn; a mutation at D279 to the amino acids: Gly, Cys, Thr, Ser,Val, Pro, Ala, or Gln; a mutation at Y282 to the amino acids: Arg, Lys,or His; a mutation at Q303 to the amino acids: Met, Pro, or Leu; amutation at Y348 to the amino acids: His or Trp; a mutation at E350 tothe amino acids: Lys, Arg, His, Tyr, Gln, Asn, Gly, Cys, Thr, Ser, Val,Pro, or Ala; a mutation at Y402 to the amino acids: Phe or Trp; amutation at D405 to the amino acids: Gly, Cys, Thr, Ser, Val, Pro, orAla; and a mutation at R406 to the amino acids: Gly, Cys, Thr, Ser, Val,Pro, Ala, Glu, Asp, Gln, or Asn.
 5. The EndoS mutant enzyme according toclaim 4, wherein the additional mutation is at least one selected fromthe following group: a mutation at H122 to Ala (H122A) or Phe (H122F); amutation at P184 to Gln (P184Q); a mutation at D279 to Ser (D279S) orGln (D279Q); a mutation at Y282 to Arg (Y282R); a mutation at Q303 toLeu (Q303L); a mutation at Y348 to His (Y348H); a mutation at E350 toAla (E350A), Asn (E350N), Asp (E350D), or Gln (E350Q); a mutation atY402 to Phe (Y402F) or Trp (Y402W); a mutation at D405 to Ala (D405A);and a mutation at R406 to Gln (R406Q).
 6. The EndoS mutant enzymeaccording to claim 5, comprising at least one mutation selected from thegroup consisting of Q303L, E350A, E350N, E350D, E350Q, and D405A as theadditional mutation.
 7. The EndoS mutant enzyme according to claim 6,wherein the additional mutation is Q303L, E350A, E350N, E350D, E350Q,D405A, H122A/Q303L, H122F/Q303L, P184Q/Q303L, D279Q/Q303L, D279S/Q303L,Y282R/Q303L, Q303L/Y348H, Q303L/E350A, Q303L/E350N, Q303L/E350D,Q303L/E350Q, Q303L/Y402F, Q303L/Y402W, Q303L/D405A, Q303L/R406Q,H122A/E350A, H122F/E350A, P184Q/E350A, D279Q/E350A, D279S/E350A,Y282R/E350A, Y348H/E350A, E350A/Y402F, E350A/Y402W, E350A/D405A,E350A/R406Q, H122A/E350N, H122F/E350N, P184Q/E350N, D279Q/E350N,D279S/E350N, Y282R/E350N, Y348H/E350N, E350N/Y402F, E350N/Y402W,E350N/D405A, E350N/R406Q, H122A/E350D, H122F/E350D, P184Q/E350D,D279Q/E350D, D279S/E350D, Y282R/E350D, Y348H/E350D, E350D/Y402F,E350D/Y402W, E350D/D405A, E350D/R406Q, H122A/E350Q, H122F/E350Q,P184Q/E350Q, D279Q/E350Q, D279S/E350Q, Y282R/E350Q, E350Q/Y348H,E350Q/Y402F, E350Q/Y402W, E350Q/D405A, E350Q/R406Q, H122A/D405A,H122F/D405A, P184Q/D405A, D279Q/D405A, D279S/D405A, Y282R/D405A,Y348H/D405A, or D405A/R406Q.
 8. The EndoS mutant enzyme according toclaim 7, wherein the additional mutation is Q303L, E350A, E350N, E350D,E350Q, D405A, Q303L/E350A, Q303L/E350N, Q303L/E350D, Q303L/E350Q,Q303L/D405A, E350A/D405A, E350N/D405A, E350D/D405A, or E350Q/D405A. 9.The EndoS mutant enzyme according to claim 4, wherein the additionalmutation is H122A, H122F, P184Q, D279Q, D279S, Y282R, Y348H, Y402F,Y402W, or R406Q.
 10. The EndoS mutant enzyme according to claim 1,wherein the activity on the N297-linked sugar chain keeps a hydrolysisrate of 50% or less in a hydrolysis reaction in a reaction solution atpH 7.4 after 24 hours.
 11. The EndoS mutant enzyme according to claim 1,wherein the EndoS mutant enzyme further exhibits an activity on theN297-linked sugar chain leading to a percent transglycosylation ofapproximately 60% or more after 48 hours of a transglycosylationreaction with 5 equivalents of a sugar donor in a reaction solution atpH 7.4.
 12. A polynucleotide encoding an EndoS mutant enzyme accordingto any one of claims 1 to
 11. 13. A vector comprising a nucleotidecomplementary to the polynucleotide according to claim
 12. 14. A hostcell transformed with a vector according to claim
 13. 15. A method ofproduction of a molecule comprising a sugar-remodeled Fc region,comprising reacting a molecule comprising an Fc region of IgG having acore GlcNAc optionally with fucose addition as an N297-linked sugarchain with a sugar donor having a structure comprising an oxazolinatedGlcNAc in the presence of an EndoS mutant enzyme according to any one ofclaims 1 to 11 to produce a molecule comprising an Fc region with aN297-linked sugar chain having a structure in which a sugar chainpossessed by the sugar donor is transferred to the core GlcNAc of theN297-linked sugar chain.
 16. The method of production according to claim15, wherein the molecule comprising an Fc region is an IgG monoclonalantibody.